IsoAmp tHDA Kit Guidelines for Optimization
PRIMER DESIGN AND AMPLICON SELECTION
HDA primers can be designed using the Primer3 program found at:http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
Product size: 70 - 120 bp
Primer size: 20 - 35 bases; optimum = 25 - 27 bases
Primer Tm: 60°C - 80°C; optimum = 68 - 72°C
Primer GC %: 30 - 60%
For other parameters use the default values in Primer3.
1) Amplicons (regions to be amplified) containing a G + C content of approximately 40% are preferable.
2) To obtain ideal amplification performance, test several amplicon regions for each target gene and design several primer sets for each selected region. The efficiency of the tHDA reactions may vary dramatically for different amplicons as well as for different primer sets within the specific region. Using primers optimized for PCR reactions may not guarantee similar performance in HDA reactions.
3) Once a set of primers yields a positive amplification result, serial primer sets longer or shorter than this set can be analyzed to generate optimal primers for increased amplification efficiency.